This research was supported by the Guangdong Science and Technology Plan Project (grant number: 2021A1515410004) and the National Key Clinical Discipline (grant number: [2012]649).

The protocol was carried out in accordance with the tenets of the Declaration of Helsinki and approved by the Ethics Review Committee of the Sixth Affiliated Hospital of Sun Yat-sen University.
The patients and families were informed of the purpose of shooting and making the surgical video, and informed consent was obtained.
1. Patient data and grouping
NOTE: From January 2018 to June 2020, laparoscopic incisional hernia re...

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hernia repair.</a> Chinese Journal of General Surgery. 31 (4), 474-480 (2022).</li><li>Fu-Xin, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Botulinum+toxin+A+facilitated+laparoscopic+repair+of+complex+ventral+hernia.">Botulinum toxin A facilitated laparoscopic repair of complex ventral hernia.</a> Frontiers in Surgery. 8, 803023 (2022).</li><li>Lihong, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observer's+assessment+of+alertness/sedation-based+titration+reduces+propofol+consumption+and+incidence+of+hypotension+during+general+anesthesia+induction:+A+randomized+controlled+trial.">Observer's assessment of alertness/sedation-based titration reduces propofol consumption and incidence of hypotension during general anesthesia induction: A randomized controlled trial.</a> Science 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Society (IEHS)) - Part A.</a> Surgical Endoscopy. 33 (10), 3069-3139 (2019).</li><li>Petersen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+prosthesis+infection+in+incisional+hernia+repair:+Predictive+factors+and+clinical+outcome.">Deep prosthesis infection in incisional hernia repair: Predictive factors and clinical outcome.</a> The European Journal of Surgery. 167 (6), 453-457 (2001).</li><li>Montgomery, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+replacement+of+an+infected+synthetic+by+a+biological+mesh+in+abdominal+wall+hernia+repair.">Evidence for replacement of an infected synthetic by a biological mesh in abdominal wall hernia repair.</a> Frontiers in Surgery. 2, 67 (2015).</li><li>Mathes, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesh+fixation+techniques+in+primary+ventral+or+incisional+hernia+repair.">Mesh fixation techniques in primary ventral or incisional hernia repair.</a> The Cochrane Database of Systematic Reviews. 5 (5), (2021).</li><li>Rieder, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesh+fixation+in+laparoscopic+incisional+hernia+repair:+Glue+fixation+provides+attachment+strength+similar+to+absorbable+tacks+but+differs+substantially+in+different+meshes.">Mesh fixation in laparoscopic incisional hernia repair: Glue fixation provides attachment strength similar to absorbable tacks but differs substantially in different meshes.</a> Journal of the American College of Surgeons. 212 (1), 80-86 (2011).</li><li>Stoikes, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomechanical+evaluation+of+fixation+properties+of+fibrin+glue+for+ventral+incisional+hernia+repair.">Biomechanical evaluation of fixation properties of fibrin glue for ventral incisional hernia repair.</a> Hernia. 19 (1), 161-166 (2015).</li><li>Bansal, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+prospective+randomized+study+comparing+suture+mesh+fixation+versus+tacker+mesh+fixation+for+laparoscopic+repair+of+incisional+and+ventral+hernias.">A prospective randomized study comparing suture mesh fixation versus tacker mesh fixation for laparoscopic repair of incisional and ventral hernias.</a> Surgical Endoscopy. 25 (5), 1431-1438 (2011).</li><li>Eriksen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pain,+quality+of+life+and+recovery+after+laparoscopic+ventral+hernia+repair.">Pain, quality of life and recovery after laparoscopic ventral hernia repair.</a> Hernia. 13 (1), 13-21 (2009).</li><li>Bageacu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laparoscopic+repair+of+incisional+hernia:+A+retrospective+study+of+159+patients.">Laparoscopic repair of incisional hernia: A retrospective study of 159 patients.</a> Surgical Endoscopy. 16 (2), 345-348 (2002).</li><li>Taylor, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laparoscopic+inguinal+hernia+repair+without+mesh+fixation,+early+results+of+a+large+randomised+clinical+trial.">Laparoscopic inguinal hernia repair without mesh fixation, early results of a large randomised clinical trial.</a> Surgical Endoscopy. 22 (3), 757-762 (2008).</li><li>Baker, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+re-operation+rate+for+recurrence+after+defect+closure+in+laparoscopic+ventral+hernia+repair+with+a+permanent+tack+fixated+mesh:+A+nationwide+cohort+study.">Decreased re-operation rate for recurrence after defect closure in laparoscopic ventral hernia repair with a permanent tack fixated mesh: A nationwide cohort study.</a> Hernia. 22 (4), 577-584 (2018).</li><li>Beldi, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesh+shrinkage+and+pain+in+laparoscopic+ventral+hernia+repair:+A+randomized+clinical+trial+comparing+suture+versus+tack+mesh+fixation.">Mesh shrinkage and pain in laparoscopic 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Laparoscopic incisional hernia repair is primarily performed using the IPOM method5, for which the placement and fixation of the mesh are key to achieving good outcomes. If the placement and fixation of the mesh are improper, the mesh will not adhere tightly to the abdominal wall and may be wrinkled or displaced. Improper mesh fixation is associated with seroma formation, abdominal infection, chronic pain, and hernia recurrence. Specifically, the treatment for mesh infections involves puncture and...

Incisional hernia is a common complication after abdominal surgery and can be properly treated only with surgery1. Compared with traditional open incision herniorrhaphy, laparoscopic herniorrhaphy has the advantages of less surgical trauma, a lower infection rate, and faster postoperative recovery2,3. Currently, laparoscopic herniorrhaphy is the method of choice for the treatment of incisional hernia if there are no contraindications4.
However, laparoscopic herniorrhaphy is technically complex. Intraperitoneal onlay mesh (IPOM) is commonly used in laparoscopic incisional hernia repair, and this involves placing a mesh into the abdominal cavity laparoscopically to cover the hernia defect5. The mesh is a new type of medium-weight monofilament polypropylene mesh covered with a hydrogel barrier on the visceral side6. For laparoscopic incisional hernia repair using the IPOM method, it is necessary to master the placement of the trocars, the techniques to separate intra-abdominal adhesions, the techniques for suturing the incisional hernia, and the methods for placing and fixing the mesh in the abdominal cavity. In particular, if the mesh is not properly placed and fixed, this can result in the recurrence of the hernia, as well as potentially serious complications such as intestinal obstruction and mesh infection7,8. Therefore, mastering the correct mesh fixation technique is an important criterion for achieving a good surgical outcome. 
The traditional method of mesh fixation for incisional hernia is to fix the mesh with a double-ring hernia nail. After the mesh is placed into the abdominal cavity, the edge of the mesh is fixed with a nail gun first, and then the edge of the hernia ring is fixed9. However, this method has poor spatial positioning, and the mesh is prone to displacement, leading to hernia recurrence. By reviewing and analyzing the various mesh fixation methods, a new "contraposition and alignment" method for mesh fixation is proposed and presented in this protocol10. In this method, the size and scope of the incisional hernia are measured in advance, after which the mesh fixation points can be marked in advance. When the mesh is placed into the abdominal cavity during the operation, nail gun fixation and suture fixation can be performed according to the previously marked locations. This method can reduce the difficulty of the operation, the operation time, the medical cost, and the occurrence of complications. In this study, this new method is compared with the conventionally used double-loop hernia nail fixation method for mesh fixation during laparoscopic incisional hernia repair surgery.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-0 Stratafix Symmetric PDS Plus Violet 45cm PS-1&#160;</td>
			<td>ETHICON</td>
			<td>sxpp1a401</td>
			<td>STRATAFIX Symmetric PDS Plus</td>
		</tr>
		<tr>
			<td>3-0 VICRYL suture</td>
			<td>ETHICON</td>
			<td>VCP316</td>
			<td>absorbable suture</td>
		</tr>
		<tr>
			<td>AbsorbaTack Fixation</td>
			<td>Covidien llc</td>
			<td>ABSTACK15</td>
			<td>absorbable nail gun</td>
		</tr>
		<tr>
			<td>Laparoscopic needle holder</td>
			<td>KARL-STORZ</td>
			<td>26173KL</td>
			<td>needle holder</td>
		</tr>
		<tr>
			<td>Laparoscopic separating forceps</td>
			<td>KARL-STORZ</td>
			<td>38651ON</td>
			<td>separating forceps</td>
		</tr>
		<tr>
			<td>Laparoscopic system (OTV-S400)</td>
			<td>Olympus</td>
			<td>CLV-S400_WA4KL530</td>
			<td>4K HD image large screen surgical laparoscope</td>
		</tr>
		<tr>
			<td>ProTack Fixation</td>
			<td>Covidien llc</td>
			<td>174005</td>
			<td>Non absorbable nail gun</td>
		</tr>
		<tr>
			<td>VENTRALIGHT ST</td>
			<td>BARD</td>
			<td>5954810</td>
			<td>Biological anti-adhesion mesh</td>
		</tr>
	</tbody>
</table>

application of a new mesh fixation method in laparoscopic incisional hernia repair

Laparoscopic incisional hernia repair using intraperitoneal onlay mesh (IPOM) is one of the most widely used minimally invasive methods for repairing incisional hernias. The laparoscopic IPOM involves implanting the mesh into the abdominal cavity through laparoscopy to repair an abdominal wall hernia. In the IPOM surgery, after the closure of the hernia ring, an anti-adhesion mesh is placed laparoscopically. The correct placement of this mesh is critical to the success of the method, and surgical skills are required to achieve perfect placement. If the mesh placement is not mastered properly, the operation and anesthesia time will be prolonged. In addition, improper placement of the mesh can lead to serious consequences, such as intestinal obstruction and mesh infection. A "contraposition and alignment" mesh fixation method is described in this study, which involves pre-marking the fixation position of the mesh to reduce the difficulty of mesh placement. A properly placed mesh is completely flat on the peritoneum, the edges are not curled or wrapped, and the mesh is adhered firmly such that there is no displacement after removing the pneumoperitoneum pressure. The "contraposition and alignment" mesh fixation technique offers the advantages of reliable placement of the mesh and fewer complications than other techniques, and it is easy to learn and master. It also allows for positioning the nail gun in advance based on the anatomy of the incisional hernia. This enables the use of the minimum number of nails possible while still ensuring good fixation, which can reduce the occurrence of complications and reduce the cost of surgery. Thus, the mesh fixation method described here is highly suitable for clinical applications based on the aforementioned advantages.

Either &#34;contraposition and alignment&#34; mesh fixation (experimental group) or traditional double-loop hernia nail fixation (control group) was performed for the patients in the study, with 42 patients in each group.
During the hernia repair surgery performed in this study, the anti-adhesion mesh was placed after the hernia ring was sutured. In the experimental group, the &#34;contraposition and alignment&#34; method was used to place the mesh, while the traditional double-loop fixation m...

Watch this Scientific Journal Video about Application of a New Mesh Fixation Method in Laparoscopic Incisional Hernia Repair at JoVE.com

Application of a New Mesh Fixation Method in Laparoscopic Incisional Hernia Repair

Presented here is a method for improving the mesh placement in laparoscopic incisional hernia repair, which can shorten the time required for mesh fixation and reduce the occurrence of postoperative chronic pain.

Laparoscopic incisional hernia repair using intraperitoneal onlay mesh (IPOM) is one of the most widely used minimally invasive methods for repairing ...

This protocol provides a new method to effectively solve the problem of mesh fixation in laparoscopic incisional hernia repair. The advantages of this method include accurate mesh positioning, firm fixation, and easy learning, especially for beginners. After mastering this method, the time of mesh fixation during operation can be significantly reduced.Text and picture demonstration cannot effectively show the whole process of this mesh fixing method, hence, visual demonstration enables understanding all of the details of this method. Begin by selecting an appropriate size anti-adhesion mesh, measure and mark the approximate range of the incisional hernia on the abdominal wall surface with a sterile ruler and marking pen. The size of the hernia ring defect can also be measured by a pre-operative CT examination.Place the ruler parallel to the longitudinal axis of the hernia defect and measure the maximum longitudinal length of the defect. Select an appropriately sized anti-adhesion mesh according to the size of the hernia ring defect. Ensure that the coverage of the mesh exceeds the edge of the defect by at least five centimeters.Mark the longitudinal length of the defect on the mesh and mark the nail gun fixation points at intervals of five centimeters on the longitudinal axis. Then extend the fixation points more than five centimeters along the marked line to the edge of the mesh which pertains to the alignment. Mark the nail gun fixation points evenly every two to three centimeters along the edge of the mesh which pertains to the contraposition.Ensure that the fixing points of the nail gun are uniformly marked two centimeters from the longitudinal axis of the defect on both sides with an interval of three centimeters. Roll the mesh such that the anti-adhesion surface will face the abdominal wall and place the rolled mesh into the abdominal cavity through the 12-millimeter puncture hole. Then unfold the mesh under laparoscopic guidance.Ensure that the marked line of the unrolled mesh overlaps the longitudinal axis of the hernia ring defect. Fix the marked points on the longitudinal axis of the mesh to the abdominal wall with non-absorbable nails using a nail gun. Fix the edge of the mesh to the abdominal wall along the marked points on the edge of the mesh with non-absorbable nails using a nail gun.Fix the mesh on the abdominal wall along the marked points on both sides of the longitudinal axis of the mesh with absorbable nails using a nail gun. The contraposition and alignment mesh fixation or traditional double-loop hernia nail fixation was performed in the experimental and control groups respectively with 42 patients in each group. The mean mesh placement time for the experimental group was approximately 32.5 minutes which was notably lower than the control group, having a mesh placement time of about 44.7 minutes.There were only two cases of chronic pain in the experimental group, significantly lower than the eight cases observed in the control group. However, there were no significant differences between the two groups in terms of seroma formation, mesh infection, recurrence of incisional hernia, the length of the post-operative hospital stay, and hospitalization expenses. Ensure that the mesh placement is accurate with adequate coverage and that precise fixation methods have been used.The application of the contraposition and alignment mesh fixation method is not limited to incisional hernia repair, but can also be used for laparoscopic parastomal hernia mesh repair and laparoscopic esophageal hiatal hernia mesh repair.

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Research

JoVE Journal

Medicine

Rapid Point-of-Care Assay of Enoxaparin Anticoagulant Efficacy in Whole Blood

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the low-molecular-weight-heparin (LMWH), enoxaparin. There are also urgent clinical situations where it would be important if this could be determined rapidly. The present assay is designed to accomplish this. We only assayed human blood samples that were spiked with known concentrations of enoxaparin. The essential feature of the present assay is the quantification of the efficacy of enoxaparin in a patient's blood sample by degrading it to complete inactivity with heparinase. Two blood samples were drawn into Vacutainer tubes (Becton-Dickenson; Franklin Lakes, NJ) that were spiked with enoxaparin; one sample was digested with heparinase for 5 min at 37 &deg;C, the other sample represented the patient's baseline anticoagulated status. The percent shortening of clotting time in the heparinase-treated sample, as compared to the baseline state, yielded the anticoagulant contribution of enoxaparin. We used the portable, battery operated Hemochron 801 apparatus for measurements of clotting times (International Technidyne Corp., Edison, NJ). The apparatus has 2 thermostatically controlled (37 &deg;C) assay tube wells. We conducted the assays in two types of assay cartridges that are available from the manufacturer of the instrument. One cartridge was modified to increase its sensitivity. We removed the kaolin from the FTK-ACT cartridge by extensive rinsing with distilled water, leaving only the glass surface of the tube, and perhaps the detection magnet, as activators. We called this our minimally activated assay (MAA). The use of a minimally activated assay has been studied by us and others. 2-4 The second cartridge that was studied was an activated partial thromboplastin time (aPTT) assay (A104). This was used as supplied from the manufacturer. The thermostated wells of the instrument were used for both the heparinase digestion and coagulation assays. The assay can be completed within 10 min. The MAA assay showed robust changes in clotting time after heparinase digestion of enoxaparin over a typical clinical concentration range. At 0.2 anti-Xa I.U. of enoxaparin per ml of blood sample, heparinase digestion caused an average decrease of 9.8% (20.4 sec) in clotting time; at 1.0 I.U. per ml of enoxaparin there was a 41.4% decrease (148.8 sec). This report only presents the experimental application of the assay; its value in a clinical setting must still be established.

Demonstration of a rapid quantitative assay of the inhibition of blood coagulation by the low-molecular-weight-heparin, enoxaparin. The contribution of enoxaparin is assessed by removing its influence through digestion with heparinase. A fuller description of the assay is detailed in our published paper.1 The assay still requires clinical confirmation.

There is the need for a clinical assay to determine the extent to which a patient's blood is effectively anticoagulated by the ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

A New Murine Model of Endovascular Aortic Aneurysm Repair

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of Abdominal aortic aneurysm (AAA) pathophysiology. There is no abdominal aortic aneurysm endograft exclusion model cheap and reproducible, which would allow deep investigations of AAA before and after treatment. We hereby describe how to induce, and then to exclude with a covered coronary stentgraft an abdominal aortic aneurysm in a rat. The well known elastase induced AAA model was first reported in 19901 in a rat, then described in mice2. Elastin degradation leads to dilation of the aorta with inflammatory infiltration of the abdominal wall and intra luminal thrombus, matching with human AAA. Endovascular exclusion with small covered stentgraft is then performed, excluding any interactions between circulating blood and the aneurysm thrombus. Appropriate exclusion and stentgraft patency is confirmed before euthanasia by an angiography thought the left carotid artery. Partial control of elastase diffusion makes aneurysm shape different for each animal. It is difficult to create an aneurysm, which will allow an appropriate length of aorta below the aneurysm for an easy stentgraft introduction, and with adequate proximal and distal neck to prevent endoleaks. Lots of failure can result to stentgraft introduction which sometimes lead to aorta tear with pain and troubles to stitch it, and endothelial damage with post op aorta thrombosis. Giving aspirin to rats before stentgraft implantation decreases failure rate without major hemorrhage. Clamping time activates neutrophils, endothelium and platelets, and may interfere with biological analysis.

Histological and biochemical modifications after aneurysm endograft exclusion are still unclear. We describe a new model of endograft implantation on a murine aneurysm. Through thrombus analysis with and without persistant circulating blood stream interface, and new endovascular biomaterials evaluation, this model will help to better understand endovascular aneurysm exclusion pathobiology.

Endovascular aneurysm exclusion is a validated technique to prevent aneurysm rupture. Long-term results highlight technique limitations and new aspects of ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

The protocol describes a new method to disaggregate human tissues and to create autologous micro-grafts that, combined with collagen sponges, give rise to human bio-complexes ready to use in the treatment of skin lesions. Further, this system preserves cell viability of micro-grafts at different times after mechanical disaggregation.

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Transvaginal Mesh Insertion in the Ovine Model

This protocol describes mesh insertion into the rectovaginal septum in sheep using a single vaginal incision technique, with and without the trocar-guided insertion of anchoring arms. Parous sheep underwent the dissection of the rectovaginal septum, followed by the insertion of an implant with or without four anchoring arms, both designed to fit the ovine anatomy. The anchoring arms were put in place using a trocar and an "outside-in" technique. The cranial arms were passed through the obturator, gracilis, and adductor magnus muscles. The caudal arms were fixed near the sacrotuberous ligament, through the coccygeus muscles. This technique allows for the mimicking of surgical procedures performed in women suffering from pelvic organ prolapse. The anatomical spaces and elements are easily identified. The most critical part of the procedure is the insertion of the cranial trocar, which can easily penetrate the peritoneal cavity or the surrounding pelvic organs. This can be avoided by a more extensive retroperitoneal dissection and by guiding the trocar more laterally. This approach is designed only for experimental testing of novel implants in large animal models, as trocar-guided insertion is currently not used clinically.

This protocol describes mesh implantation in the ovine rectovaginal septum using a single vaginal incision technique, with and without the trocar-guided insertion of anchoring arms.

This protocol describes mesh insertion into the rectovaginal septum in sheep using a single vaginal incision technique, with and without the trocar-guided ...

Laparoscopic Repair of Para-Esophageal Hernia Using Absorbable Biosynthetic Mesh

Para-esophageal hernia repairs are challenging procedures and there is no consensus on the optimal approach to repair. Mesh reinforcement has been associated with lesser hernia recurrence when compared to the primary suture repair. The type of mesh that is most appropriate is still debatable. Synthetic and biosynthetic materials have been studied in the literature. It is well documented that a synthetic mesh is associated with esophageal erosion and migration into the stomach. Though there are limited long-term data on biosynthetic mesh, the short-term results are excellent and promising.
This paper illustrates how a biosynthetic prosthesis can be safely used with fibrin glue fixation and anterior Dor fundoplication to repair any para-esophageal defect. The absorbable biosynthetic mesh has been shown to produce good long-term patient satisfaction outcomes and low recurrence rates compared to conventional methods including repair with synthetic mesh. This technique also avoids the risk of esophageal erosion whilst strengthening the repair. Tacks that are still widely used to secure the mesh can be abandoned due to the associated risk of developing cardiac tamponade, or other disastrous consequences. This repair method, also, highlights how the prosthesis can be fashioned into a V-shape and easily placed in an onlay fashion behind the esophagus. The protocol demonstrates an alternative and safer method for mesh fixation using fibrin glue.

Presented here is a protocol of para-esophageal hernia repair. Use of absorbable biosynthetic mesh avoids the risk of erosion through the esophagus whilst reinforcing the repair. Glue fixation is preferred to avoid the risk of trauma such as bleeding or cardiac tamponade, which are associated with stitches or tacks.

Para-esophageal hernia repairs are challenging procedures and there is no consensus on the optimal approach to repair. Mesh reinforcement has been ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Laparoscopic Non-Mesh Cerclage Pectopexy for Pelvic Organ Prolapse

Pelvic organ prolapse (POP) is widespread among the female population and significantly impairs the patient's quality of life. It is important to restore apical support for treating POP. Sacrocolpopexy and pectopexy are indicated for apical prolapse. Using a synthetic mesh in these techniques increases success by enhancing apical support. However, the implantation of synthetic mesh is associated with mesh-related complications. In addition, the exorbitant cost of synthetic mesh and lack of universal access limit the popularity of these procedures. The current study develops a unique technique known as laparoscopic non-mesh cerclage pectopexy (LNMCP), in which permanent cervical cerclage sutures are embedded in the round ligament until the iliopectineal ligament. The iliopectineal ligament was sutured, resulting in a firm cervical suspension. The procedure was successfully performed in 16 cases in the hospital. The surgical duration was 67.8 min &plusmn; 15.5 min, and the blood loss was 73.1 mL &plusmn; 51.1 mL. No procedural complications were seen. LNMCP is associated with an objective success rate of 100% and a subjective success rate of 93.8%. LNMCP for patients with apical prolapse obviates the need for a mesh, thereby avoiding complications associated with mesh erosion and reducing medical costs. In addition, it is easy to perform even in resource-poor areas without access to synthetic mesh.

The present pilot study describes the development of laparoscopic non-mesh cerclage pectopexy to treat pelvic organ prolapse. The procedure can be used to prevent any complications associated with the use of mesh.

Pelvic organ prolapse (POP) is widespread among the female population and significantly impairs the patient's quality of life. It is important to restore ...